Dendritic cells constitute a distinct group of potent antigen presenting cells (APC) which are bone marrow derived and found as trace populations in the circulation as well as within both lymphoid and nonlymphoid tissues (1-3). Although their importance as the most effective haemopoietic cell involved in the initiation of primary immune responses has been well demonstrated (4-7), no human dendritic cell specific lineage marker has been identified and most features of their ontogeny and relationship to other leukocytes remains unclear.
Phenotypically, human dendritic cells are characterised (1-3,7-11) by a high density of class II MHC antigens, the presence of a wide range of adhesion molecules and the absence or low expression of a range of lineage specific cell surface antigens (CD3, CD14, CD16, CD19, CD57). A number of activation antigens including IRAC (12), HB15 (13), 4F2 (8), IL-2R (7,8), and B7/BB-1 (7,14) have also been reported on human dendritic cells, particularly after activation, although the anti-IRAC and HB15 reagents have not been shown to stain isolated fresh blood dendritic cells. Despite this phenotypic characterisation, identification and therefore purification of dendritic cells remains difficult as the majority of these antigens are expressed by other resting and activated cell types. Many of the functional and phenotypic features of dendritic cells are shared by both Hodgkins cells (HC) and Hodgkins Disease (HD) derived cell lines and there is increasing evidence to support the hypothesis, that in some instances, HC represent a malignant form of dendritic cell (15-17).
It is therefore an object of the present invention to inter alia provide a method of generating immunological reagents for use in a process for identifying and purifying dendritic cells.
Previous attempts to isolate antibodies specific for human dendritic cells have been largely unsuccessful. A number of these attempts have been directed toward monoclonal antibodies and have utilised an improved dendritic cell purification method to prepare immunogens (32) and a rapid two-colour flow cytometric screening procedure that allows large numbers of hybridoma supernatants to be examined in each fusion (33). These attempts failed, yielding only hybridomas that bind common antigens of both dendritic cells and other leukocytes.
Further attempts to generate dendritic cell-specific antibodies, in this case, through tolerisation of the host prior to immunisation with dendritic cells, have been reported (34). Two methods of tolerisation are compared. The first method involves the administration of a cytotoxic antiproliferative agent (cyclophosphamide(CP)) in conjunction with immunodominant leukocyte antigens to preferentially kill B cells that respond to unwanted epitopes. In the second method, tolerisation is induced by injecting the undesired immunodominant leukocyte antigens during the neonatal period.
It is reported that the first method generates only non-specific immunosuppression whereas the second method potentially leads to a state of antigen-specific non-responsiveness. However, the ability of the tolerised host to generate antibodies specific for dendritic cells is not demonstrated nor is any data provided regarding the efficiency of the method in producing a positive serological response to dendritic cells. All that is shown is tolerisation ie. lack of reactivity with T blasts.
The applicant's own investigations have however found that, in practice, an approach involving tolerisation of a neonate host with available immunodominant leukocyte antigens is not generally effective in raising immunologically useful antibodies against dendritic cells. In particular, the applicants attempts to tolerise neonatal mice with leukocyte antigen expressed on Epstein Barr Virus (EBV) transformed lymphoblastoid cell lines and to then raise specific antibodies against dendritic cells were unsuccessful.
This lack of success in generating dendritic cell-specific antibodies using the above approach is consistent with anecdotal reports of similar unsuccessful attempts made by other workers in this area.
Surprisingly, the applicants further investigations have found that when some monocytoid cell lines are used as the tolerising antigen in relation to a neonatal host, subsequent immunisation with dendritic cells or Hodgkins Disease derived cell lines does lead to the generation of antigen-specific antibodies. It is in part to this unexpected finding that the present invention is directed.